In Vivo Processing Assay Based on a Dual-Luciferase Reporter System to Evaluate DROSHA Enzymatic Activity - miRNA Maturation: Methods and Protocols - page 87

6. Four days after transfection proceed with in vivo assay ( see
Note 4 ). Complexes do not have to be removed following
transfection ( see Note 5 ).

1. One day before transfection seed the cells into 12-well plates.
Cell density should reach 50-80 % confluence on the day of
transfection.
2. On the day of transfection, replace the medium with 1 mL of
complete growth medium.
3. For each well to be transfected prepare transfection mix by
mixing 200 pRL-CMV + 100_ng pMirLuc16-2ss + 1 μg_
pSuper-shEGFP in 93.5 μL serum-free DMEM ( see Note 6 ).
Add 4 μL TransIT-LT1 transfection reagent, mix well, and
incubate for 20 min at room temperature.
4. Dropwise add 100 μL of transfection mix to the cells and shake
gently by rocking the plate back and forth.
5. Lyse cells and assay luciferase signal 18-24 h after
transfection.

3.1.2 In Vivo Assay
Transfection

1. Prepare proper amount of 1× passive lysis buffer by diluting
one part of 5× passive lysis buffer (keep on ice) in four parts of
ddH 2 O ( see Note 7 ).
2. Gently remove the medium from the cell culture wells and
rinse cells with 500 μL 1× PBS.
3. Add 100 μL of 1× passive lysis buffer and shake at room tem-
perature for 10 min.
4. Transfer the lysate to a 1.5-mL microcentrifuge tube, and spin
down cells at 16,000 × g at room temperature for 1 min to
remove cell debris.
5. Transfer supernatant into a new 1.5-mL microcentrifuge tube,
and keep the samples on ice before measurement ( see Note 8 ).
6. Pipet 15 μL of each sample into 96-well plate ( see Note 9 ).

3.2 Cell Lysis

3.3 Measurement
of Luciferase Activity

1. Thaw proper amount of buffers referring to 100 μL buffer/
well. Let warm to room temperature. Always mix the reagent
prior to use as thawing generates density and composition gra-
dients ( see Notes 10 and 11 ).
2. For measurement a luminometer can be used with two auto-
matic injectors. The following parameters are set in our instru-
ment settings: injection volume of buffer 75 μL with 5-s delay
between injection and measurement ( see Note 12 ).
3. Depending on the luminometer used it might be necessary to
prime auto-injector systems with buffers before measurement.
4. Record the firefly luciferase activity measurement by adding FF
buffer to the well.